A review of the correlation of tergites, sternites, and leg pairs in diplopods
© Janssen et al; licensee BioMed Central Ltd. 2006
Received: 23 August 2005
Accepted: 02 February 2006
Published: 02 February 2006
In some arthropods there is a discrepancy in the number of dorsal tergites compared to the number of ventral sternites and leg pairs. The posterior tergites of the Diplopoda (millipedes) each cover two sternites and two pairs of legs. This segment arrangement is called diplosegmentation. The molecular nature of diplosegmentation is still unknown. There are even conflicting theories on the way the tergites and sternites/leg pairs should be correlated to each other. The different theories are based either on embryological analyses or on studies of the adult morphology and turned out to be not compatible with each other. We have previously used the expression patterns of segmentation genes in the pill millipede Glomeris marginata (Myriapoda: Diplopoda) to study millipede segmentation. Here we review the existing models on the alignment of tergites and leg pairs in millipedes with special emphasis on the implications the gene expression data have on the debate of tergite and leg pair assignment in millipedes. The remarkable outcome of the gene expression analysis was that (1) there is no coupling of dorsal and ventral segmentation and, importantly, that (2) the boundaries delimiting the tergites do neither correlate to the embryonic boundaries of the dorsal embryonic segments nor to the boundaries of the ventral embryonic segments. Using these new insights, we critically reinvestigated the correlation of tergites, sternites, and leg pairs in millipedes. Our model, which takes into account that the tergite boundaries are different from the dorsal embryonic segment boundaries, provides a solution of the problem of tergite to sternite/leg pair correlation in basal milipedes with non-fused exoskeletal elements and also has implications for derived species with exoskeletal rings. Moreover, lack of coupling of dorsal and ventral segmentation may also explain the discrepancy in numbers of dorsal tergites and ventral leg pairs seen in some other arthropods.
A model for tergite-sternite-leg pair assignment based on adult ring-forming millipedes
Fortunately, however, there are other millipede species, e.g. the members of Juliformia (Fig. 2C) that have their tergites and sternites fused together into rigid armour rings (for an overview see [1, 2]). Each pair of legs is jointed to a sternite that is fused to a tergite via the lateral pleurites, in this way making leg pair/tergite alignment seemingly straightforward and unambiguous. In these animals, the first tergite (collum) is free (i.e. not fused to any ventral component). The second tergite is fused with the sternites of the first pair of legs into the first cuticular ring (ring 1) (see Fig. 2C). The third and fourth tergites are fused with the second and third trunk sternites, respectively, thus forming ring 2 and 3. The anterior part of the juliform trunk, therefore, consists of a free collum, followed by three rigid rings, each of which is bearing a single pair of legs. Posterior to this something surprising happens: each tergite is fused not to a single sternite, but to a pair of sternites. The result: starting with ring 4 each ring has two pairs of legs (see Fig. 2C).
In order to distinguish between the tergites that correlate with only one leg pair and those that correlate with two leg pairs, tergites II, III, and IV are called haplotergites, whereas all following tergites are so-called diplotergites. The phenomenon that one dorsal segmental unit (one diplotergite) is correlating with two ventral segmental units (two sternites and two leg pairs) is called diplosegmentation.
As already mentioned above, in non-ring-forming species with free tergites, pleurites and sternites, the correlation of leg pairs with tergites is unclear since they are not fused into rings. However, the results from ring-forming species can be extrapolated to non-ring-forming species, resulting in a similar assignment (Fig. 3A). Thus, according to the correlation scheme derived from the study of ring-forming millipedes, the millipede trunk has three "haplosegments" followed by a number of "diplosegments" (Fig. 3).
Another model for tergite-sternite-leg pair assignment based on embryology in non-ring-forming millipedes
Both models most significantly differ in the assignment and location of the tergites and the diplosegments, both of which are metameric structures. We decided to study the long-standing issue of the discrepancy in assignment of dorsal and ventral metameric units by analyzing genes controlling segmentation in the millipede Glomeris marginata . Our results show surprising differences in expression of these genes in dorsal and ventral metameric units. In our previous paper  we focused on the different mechanisms that must act during the formation of the dorsal and ventral metameric units in the embryo. In the present paper we discuss in detail the implications the lack of coupling of dorsal and ventral segmentation has for the debate on the correlation of tergites with sternites and leg pairs. We review here the evidence for an alternative model for tergite – sternite/leg pair assignment that provides a solution for the problems in the previous models.
Segmentation genes and segment formation in arthropods
Segmentation genes have been studied in great detail in the fruit fly Drosophila melanogaster, and as a consequence, Drosophila segmentation still forms a paradigm for segmentation. The Nobel Prize awarded D. melanogaster mutagenesis screen reported by Nüsslein-Volhard and Wieschaus  revealed several classes of mutant segmentation phenotypes. The affected genes in these mutants are the so-called segmentation genes and were shown to act in a hierarchic gene cascade to divide the early embryo into a repeating series of segmental units along the anterior posterior axis. One of the classes of segmentation genes are the segment-polarity genes that are responsible for establishing and maintaining the boundaries of the initial metameric units, the parasegments. These genes also define the A-P polarity within each metameric unit.
The best-studied segment-polarity gene is the engrailed gene. The engrailed gene is expressed in segmentally reiterated stripes in the embryo. In Drosophila, another segment-polarity gene, the wingless gene, is active in cells just anteriorly adjacent to the stripe of engrailed expressing cells. The engrailed and wingless expressing cells are mutually exclusive and define an important boundary between them, the parasegmental boundary. Genetic and molecular studies have shown that the parasegments are fundamental units in the design of the early Drosophila embryo [8–11].
The analysis of Drosophila segment-polarity gene homologs in other arthropods, like more primitive insects, crustaceans, centipedes, and spiders, revealed that the role of these genes in defining the parasegmental boundary represents a conserved function among arthropods (e.g. [12, 13]). Also in these arthropods wingless/Wnt genes are active in cells anteriorly adjacent to the engrailed expressing cells. Detailed analyses, e.g. in the spider Cupiennius salei and the centipede Lithobius atkinsoni, demonstrated that the border between the engrailed and wingless/Wnt expressing cells also defines the important boundary of the parasegment, similar as in insects [12, 13]. Also in crustaceans engrailed expression is associated with units comparable to parasegments . The expression of these genes thus forms an excellent marker for the formation of the segmental units in arthropods.
Segmentation genes in dorsal and ventral embryonic segments of the millipede Glomeris
On the dorsal side the gene expression patterns are significantly different. As described above, development of the presumptive dorsal tissue in Glomeris starts later than the ventral tissue, and is forming the so-called lateral plates [4, 6] (Fig 4). The anterior portion of the germ band up to the 4th ventral embryonic segment of the trunk initially comprises ventral tissue only. At stage 3 the development of the dorsal tissue commences (Fig 4AB) and engrailed expression is activated de novo in this tissue (Fig. 6A,B asterisks). These dorsal engrailed stripes, however, have no connection with the ventral engrailed stripes and in contrast to the ventral engrailed stripes are not associated with the morphologically visible grooves. Furthermore, the dorsal engrailed stripes are not aligned with the ventral engrailed stripes, but are shifted in relation to them. More posterior the dorsal tissue forms simultaneously with the ventral tissue. Here, the engrailed gene is expressed in each ventral embryonic segment, while the gene is only expressed in the dorsal tissue corresponding to every other ventral embryonic segment . On the dorsal side there are thus reiterated stripes of engrailed expression (Fig. 6), but they do not correspond in a one-to-one fashion to the engrailed stripes on the ventral side, they are not associated with a morphological boundary, and, surprisingly, they are not associated with wingless expression (Fig. 7) (see also ). The lack of wingless expression in the dorsal tissue implies that there cannot be an interface of engrailed and wingless expressing cells to define a boundary, like on the ventral side. This argument is strengthened by the lack of cubitus interruptus gene expression anterior to the dorsal stripes of engrailed expression . Cubitus interruptus is the activator of wingless gene expression. These data imply that the genetic interactions must be different in the dorsal and ventral tissue of Glomeris. In order to acknowledge this fact we regard the dorsal metameric units, the lateral plates , as segmental units and in order to distinguish them from the ventral embryonic segments we designate them as: dorsal embryonic segments
There thus appears to be a lack of coupling of dorsal and ventral segmentation as is obvious from several pieces of evidence. (1) The anterior dorsal tissue forms later than the ventral one and (2) the engrailed stripes appear de novo in this anterior dorsal tissue. (3) The dorsal engrailed stripes are not located near the morphological boundaries between the metameric units and (4) the dorsal metameric units differ in number from the ventral ones. (5) But most significantly, the genetic interactions must be different as wingless expression is missing in dorsal tissue. Dorsal and ventral segmentation thus are not coupled in Glomeris.
Dorsal engrailed expression coincides with tergite borders
Sternite – tergite correlation in diplopods
Our data show that the tergite borders are defined independently from the embryonic segment borders (both ventral and dorsal) and are established by a distinct genetic mechanism that uses engrailed but not wingless signalling. Thus, in terms of genetic mechanisms involved, the segmentation of the dorsal exoskeleton appears to be different from the segmentation processes leading to the formation of the ventral and dorsal embryonic segments. On the other hand, the formation of the tergites seems to build on the prior establishment of the dorsal embryonic segments. Tergite borders invaginate roughly in the middle of each dorsal embryonic segment and the tergites themselves are formed by the posterior half of one dorsal embryonic segment and the anterior half of the following one. This process bears some resemblance to resegmentation in vertebrates where the primary metameric units, the somites, are divided in the middle and the final metameric units, the vertebral bones, are formed from the posterior half of one somite and the anterior half of the next one (reviewed in ). The two processes in Glomeris and vertebrates certainly are not homologous, but might nevertheless share some developmental mechanisms by convergent evolution. These new insights now allow a modified model for tergite – sternite/leg pair correlations in basal milipedes with non-fused exoskeletal elements, like Glomeris (see Fig. 9A). But our data also have ramifications to the ring-forming process in derived millipedes. According to our results, the tergites are shifted in relation to the sternites on the ventral side of the animals. In order to fuse into a ring, a tergite therefore has to "choose" between two alternatives: for example tergite III may fuse either with the sternite a bit in front of it (sternite of ventral embryonic trunk segment 2), or with the sternite a bit behind of it (sternite of ventral embryonic trunk segment 3). The former alternative where the tergites fuse with the sternites shifted slightly in front of them would lead to the situation found in the large majority of all ring-forming species (Fig. 9B): tergites II, III, and IV fuse with sternites 1, 2, and 3 respectively, whereas all following tergites fuse with two consecutive sternites, leading to three rings with a single leg pair followed by rings with two leg pairs each. However, the second alternative also seems to be realized in nature, because there is a group of juliform millipedes, the Spirobolida, that have four single leg bearing rings . Assuming that in these animals the tergites fuse with the sternites of the slightly posterior ventral embryonic segments, the result would be four rings with a single leg pair followed by rings with two leg pairs each (Fig. 9C).
Although our results suggest a solution to the problem of tergite – sternite/leg pair correlations in diplopods, they do not provide an immediate answer as to the origin and evolution of diplosegments in this myriapod group. Our results do not support the existence of single segments with two pairs of legs each. Rather, the expression of engrailed, wingless and other segmentation genes  suggests that the ventral embryonic segments correspond to the body segments of other arthropods and these ventral embryonic segments bear only a single pair of legs each. The term "segment" is problematic when used for metameric units in millipedes, because as we have shown during embryogenesis there are at least three different series of repeated units (ventral embryonic segments, dorsal embryonic segments, tergites) all of which are established independent from each other. In the past the term "segment" has been used quite indiscriminately for all these repeated units and also for composite units like the fused armour rings in ring-forming species (for an extended discussion of arthropod segments and their homology, see Ref.  and ). Our data reject the idea that the rings in adult ring-forming species are homologous to the body segments of other arthropods. Rather, these rings seem to be a specific innovation of these diplopod species and are a composite of parts from different metameric units. In order to avoid further confusion in the future, we suggest not to use the term "segment" for the body rings and to denote the rings with a single leg pair as "haplorings" instead of "haplosegments" and the rings with two pairs of legs as "diplorings" instead of "diplosegments".
It is clear now, that the origin of diplosegmentation cannot be understood on the basis of the tergites alone, but must also take into account the origin and development of the dorsal embryonic segments, especially those that correlate with two ventral embryonic segments. Unfortunately, so far we were not able to identify genes that are expressed at the boundaries between the dorsal embryonic segments; thus at present the establishment of this boundary is unclear. Research into the developmental genetics of the dorsal embryonic segments in Glomeris is thus an exciting topic and will lead to further insights into the peculiar mode of diplosegmentation in the future.
Apart from the still unclear mode of diplosegmentation, however, our finding of the independent segmentation of the dorsal exoskeleton (tergites) in Glomeris, may explain dorsoventral peculiarities in other arthropods. As mentioned in the introduction, taxa like e.g. notostracan crustaceans, pauropods and symphylans show a mismatch of tergite numbers with leg pairs. Independent dorsal and ventral segmentation in a similar manner as in Glomeris, and the independent establishment of the tergites, in theory can lead to any combination of tergites with any number of leg pairs. If tergite formation and the formation of segments also are not coupled in notostracans, pauropods, and symphylans, this could be the cause for the dorsoventral discrepancies in these arthropods.
We would like to thank Wolfgang Dohle for stimulating discussions on Glomeris segmentation. The work of W.D. was supported in part by the DFG via SFB 572 of the University of Cologne and by the European Union via the Marie Curie Research and Training Network ZOONET (MRTN-CT-2004-005624), and the work of N.-M.P. was supported by a DFG grant (TA99/19-2).
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